Maize grain yield enhancement in modern hybrids associated with greater stalk lodging resistance at a high planting density: a case study in northeast China

Lodging resistance is a critical trait in modern maize breeding. This study aimed to examine maize stalk lodging and its related characteristics in response to increasing planting densities in modern hybrids. A two-year field trial was conducted from 2018 to 2019 with two widely grown commercial hybrids (‘Xy335’ and ‘Fm985’) and three planting density treatments of 4.5 × 104 (low density, LD), 6.5 × 104 (medium density, MD), and 8.5 × 104 plants/ha (high density, HD). New hybrid Fm985 had a significantly higher grain yield and lower lodging rate at HD, while there was no significance at LD and MD. Compared to Fm985, old hybrid Xy335 had a significantly high plant height, ear and gravity height, and culm length (CL) across the three planting densities, while opposite stalk bending strength (SBS), dry weight per unit length (DWPU), cross-sectional area, and the cellulose and lignin content in the basal internode were low. Correlation and path analysis revealed that kernel number per ear and lodging rate directly contributed to maize grain yield, while lodging-related traits of SBS, stem lignin, and DWPU had an indirect effect on maize grain yield, suggesting that modern hybrid maize yield enhancement is associated with greater stalk lodging resistance at a high planting density in northeast China.

Maize (Zea mays L.), which originated from Mesoamerica and was introduced to China approximately 500 years ago 1 , has become the first major cereals crop in China since 2012 and plays an important role in ensuring an ever-increasing food supply 1 . The Songliao Plain, located in northeast China (containing Jilin, Heilongjiang, Liaoning, and the eastern part of Inner Mongolia of China), is one of the three world-famous Black soil areas, with arable land of 5.68 × 10 6 ha and maize yield of 2.61 × 10 7 Mg in 2019, accounting for nearly a quarter of the total national grain yield and about one-third of the total national commodity grain output in China 2 . Thus, maize plays an important role in feeding the nation and is known as the "ballast stone" delivered by the first domestic "White Paper on Northeast Black Land (2020)" in China 3 .
Maize is a widely grown C4 crop with high photosynthetic activity/efficiency, leading to high grain and biomass yield potential globally 4 . The potential grain yield of maize has increased tremendously in the past several decades in China 1 , as well as in other countries such as the US 5,6 and South Africa 7 . Numerous studies have demonstrated that an increase in planting density is responsible for the increase in maize production, worldwide [8][9][10][11][12] . Lu et al. 13 reported that maize genetic gain averaged 78.99 kg −1 ha −1 year −1 from the hybrids released from 1999 to 2018 in China, with 54.41% of the total yield gain attributed to breeding. In the US, grain yield increased Figure 1. Comparison of maize grain yield, and lodging rate in 2018 (a) and 2019 (b) at varied planting densities. LD, MD, and HD represent low, medium, and high planting densities, respectively. Bars denote the SE of the mean (n = 3). ns, not significant (P > 0.05), ** and *** indicate differences of grain yield significant at 0.01and 0.001levels, # and ### indicate differences of lodging rate significant at 0.01and 0.001levels. Scientific Reports | (2022) 12:14647 | https://doi.org/10.1038/s41598-022-18908-z www.nature.com/scientificreports/ of the three PDs (Fig. 1). For hybrid Fm985, the maize grain yield increased with the increase in PD, while the highest maize yield was recorded at MD for Xy335, which indicated that newly released cultivar Fm985 had a high yield potential at HD. Maize hybrid (H), planting density (PD) and year (Y) significantly affected yield components, i.e., ear number per ha and 100-grain weight, while kernel number per ear was only significantly affected by planting density (PD) ( Tables 1, 2). Therefore, the modern commercial hybrid Fm985 had a higher ear number per ha and 100-kernel weight across the three PDs. Significant differences were observed at HD, while differences were non-significant at LD and MD for both hybrids. Kernel number per ear was significantly affected by PD, whereas it was not significantly affected by hybrid or other factors.  www.nature.com/scientificreports/ Stalk lodging rates were significantly affected by hybrid (H), planting density (PD), year (Y), and the interaction of PD × Y (Fig. 1, Table 1). The lodging rate of hybrid Xy335 was 5.26-fold higher than Fm985 in 2018, and 4.04-fold higher in 2019 across the three PDs. Among the different PDs, the average lodging rates at LD, MD, and HD were 5.21%, 12.60% and 21.71%, respectively, for Xy335, while they were 0.42%, 3.92% and 4.42%, respectively, for Fm985. The results indicated that the maize hybrid Fm985 had a higher stalk lodging resistance in the present study.
Stalk lodging-related morphological characteristics. Maize plant architecture-related traits, such as plant height (PH), ear height (EH), and gravity height (GH), were investigated, and hybrid maize Xy335 had a higher PH, EH, and GH in all tested years (Table 3). Compared with low density (LD) and medium density (MD), PH, EH, and GH at high density (HD) had significantly higher values for both cultivars, while no significant differences were observed in these plant architecture characteristics for PD and year, except for EH (Tables 1, 3).
The basal stalk morphological characteristics related to stem lodging resistance of dry weight per unit (DWPU) were significantly affected by H and PD; At LD and MD, the differences in DWPU under the same PD were not significant for either cultivars, while hybrid Fm985 had a higher DWPU than XY335 at HD in both years. Culm length (CL) and cross-sectional area (CSA) of the stem were both significantly influenced by PD, while no significant effect was observed in H, Y, and other interactions (Tables 1, 3).

Stalk bending strength (SBS) and carbohydrates content of basal internode.
Stalk bending strength (SBS) was significantly reduced with an increase in PD in both hybrids in two consecutive years (Fig. 3, Table 1). Hybrid Fm985 showed a relatively higher SBS at MD and HD, which were 4.74% and 21.02% higher than Xy335, respectively, in 2018, and 8.24% and 18.19% higher, respectively, in 2019.

Path analysis.
To analyze the causal links between maize grain yield and stem lodging-related morphological and mechanical properties, a structural equation model (SEM) approach was conducted using these characteristics. Kernel number per ear and lodging rate directly contributed to maize grain yield; maize lodging rate (p < 0.001), which was directly affected by SBS (p < 0.001) and GH, had a negative effect on 100-grain weight and kernel number per ear and an indirect effect on maize grain yield (Fig. 5). In addition, the stem lignin content directly contributed to DWPU and SBS, which were highly related to maize lodging rate and indirectly contributed to maize grain yield ( Fig. 5 and 6).

Discussion
Lodging is a serious problem that hinders grain yield stability and quality in modern crop production 8,21 . Stalk lodging is not only affected by the nature of the cultivar genotypes and agricultural practices 10 but is also associated with the growth conditions/environment, including wind speed and rainfall 27 . The Super Typhoon Lekima caused heavy rainfall in the mid grain-filling stage in the 2019, resulting in a higher lodging rate and reduction of grain yield, compared with 2018. However, hybrid Fm985 showed a higher grain yield and a lower stalk lodging rate than Xy335 in consecutive years at a high planting density. Therefore, the newly released maize hybrid Fm985 had a higher lodging resistance than the old hybrid Xy335. These results are consistent with the previous   16 and Luque et al. 24 who demonstrated that the overall trend in new cultivars is an increase in grain yield and a reduction in stalk lodging in northeast China and other places. Stalk lodging induces bending and/or breakage of stems during maize growth, generally caused by the damages to vascular bundles 28 , and thereby preventing water transport from root to leaves and impairing phloem transportation of assimilates from the mesophyll into the stalk phloem. Thus, more photoassimilates may be allocated to leaves or stalks than to the ear 2,29 , affecting grain filling and decreasing the kernel weight and effective ear number. Therefore, the decreased yield components of 100-grain weight and ear number were observed in the less lodging-resistant hybrid, Xy335, in the present study.
Maize stalk lodging resistance is also related to many plant characteristics 21 . Increasing planting density brings fiercer competition for solar radiation and the availability of other resources for individual plants, which will be shaped due to plants adaptive regulation 10   www.nature.com/scientificreports/ who found that the PH and EH were enhanced with the planting density raised from 4.5 × 10 4 to 8.0 × 10 4 plant/ ha under three N levels. Hybrids with a greater stalk height and ear position are more susceptible to lodging due to higher plant gravity and increased upper fresh weight 21 . In the present study, the maize hybrid Xy335 had a significantly higher PH, EH, and GH at a high planting density, while the differences were non-significant for hybrid Fm985. These results agree with those of previous studies of Cao et al. 8 and Xue et al. 31 who also showed that hybrid Xy335 is lodging-susceptible at a high planting density. In addition, plant morphological characteristics have evolutionarily changed with the genetic improvements in the past few decades 16 . A linear positive increase in plant height has been demonstrated with an increase in maize grain yield in the US 32 , as well as in China 1 . However, no differences in grain yield were found between the two hybrids at LD and MD, suggesting that the improved grain yield at HD is associated with enhanced stalking lodging resistance, which contributes to a small yield loss. Numerous studies have shown that the basal part of the stalk plays an important role in maintaining lodging resistance 21,33 . Stalk morphological characteristics of the basal stem i.e., internodes length, DWPU, and CSA, have a strong correlation with stem wall thickness 17,19 and therefore influence lodging susceptibility. In the present study, maize hybrid Fm985 had a higher DWPU than Xy335, while no significant differences in CL and CSA were neither found in hybrids nor in planting densities, indicating that DWPU may contribute to the higher bending strength of Fm985 at a high planting density. Previous studies have shown that basal internode DWPU and CSA are correlated with SBS and lodging resistance 8 . The DWPU of the third aboveground internode was the most important factors affecting stem lodging resistance 34 . A recent study confirmed that lodging resistance in maize genotypes had a higher DWPU 35 , which agrees with our results that the newly released maize hybrids Fm985 had a higher DWPU and stem lodging resistance than old hybrid Xy335. A high planting density caused low internode DWPU, which can be attributed to low light intensity by mutual shading 36 , as well as assimilates partitioning between the corn cob and basal stem 35 . In the present study, the newly released maize hybrids Fm985, with erect leaves, may have higher light interception and thus contribute to higher internode DWPU and stalk lodging resistance 27 . However, more possible mechanisms should be addressed.
The maize lodging resistance ability is a complicated genetic mechanism. In addition to the morphological traits of plants, the basal stem chemical composition of cellulose and lignin is also important and contributes significantly to lodging resistance 19 . Culm cellulose can determine stem strength and increase stem rigidity, while lignin, as a vital structural component of the secondary cell walls, can provide strength to plants 37 . Thus, the cellulose and lignin content in the culm plays a critical role in the stalk strength and rigidity of the basal stem, therefore influencing stalk lodging resistance 21 . Significant differences in cellulose and lignin content were observed between planting densities and hybrids. Maize hybrid Fm985 had a higher cellulose and lignin content in the basal stem, thereby providing strong bending strength and stiffness of plant culms. These results agree with Zhang et al. 38 who demonstrated that high-yield hybrids with strong stem lodging resistance have a higher structural carbohydrate contents in the basal culm of rice. Similar results were shown by Sekhon et al. 39 in maize. Moreover, transgenic technology has been used to introduce a lignin-deficient mutant gene (gh2) to normal plants and has demonstrated that culm lignin content may play a primary role in increasing stalk lodging resistance 40 . Therefore, further studies with detailed analyses will shed more light on the association between structural carbohydrate contents and stalk lodging resistance. Additionally, when the planting density increased from 45,000 (LD) to 85,000 plant/ha (HD), the cellulose and lignin content of Fm985 slightly decreased, while in Xy335, it distinctly decreased, suggesting that developing high lignin varieties may act as an crop breeding strategy to enhance crop stalk lodging resistance.
Stalk lodging occurred mainly depends on two conditions: the strength of external loading conditions (e.g., wind) and its duration 21 ; thus, lodging resistance not only depends on basal stem rigidity but is also related to the tensile strength of the plant 41 . Stalk bending strength is a measure used to estimate stalk mechanical strength, both related to stem rigidity and tensile strength 19 . The maize hybrid Fm985 showed a relatively higher SBS at MD and HD in the present study, suggesting that the improved SBS of Fm985 may contribute to its enhanced lodging resistance. Moreover, the correlation between lodging rate and related traits also showed a significant negative effect between SBS and maize stalk lodging rate, indicating that SBS after 35 days of silking could act as www.nature.com/scientificreports/ an important indicator for estimating lodging resistance in maize breeding or cultivation. SBS is highly associated with stalk morphology and the chemical characteristics of the DWPU, basal internode length, and carbohydrate contents in the culm 28,29 . The correlation analysis also confirmed that SBS is significantly positively related to DWPU (r = 0.86, p < 0.01) and stem lignin content ( r = 0.84, p < 0.01), and highly negatively correlated with EH (r = − 0.79, 0.01 < p < 0.05), and GH (r = − 0.77, 0.01 < p < 0.05). These factors may coordinately play a key role in enhancing the plant stalk lodging resistance and thereby reduce the lodging rate, in contrast to maize grain yield. Structural equation modeling (SEM) is a powerful, multivariate technique increasingly used in scientific investigations to evaluate multivariate relationships 42 . In recent years, it has been widely used to estimate the direct or indirect effects on plant traits 43,44 . Path analysis using the SEM model showed that kernel number per ear directly contributed to maize grain yield in the present study, and lodging rate was directly affected by SBS. GH had a negative effect on kernel number per ear and 100-grain weight, having an indirect effect on maize grain yield. Stem lignin content, which significantly affected DWPU and SBS, also directly contributed to the grain yield. These results are consistent with the previous studies of Xue et al. 44 and Shinoto et al. 37 , and agree with our previous study that SBS and DWPU in maize were markedly enhanced by the application of plant growth regulators (PGRs) in Northeast China 8 . In northeast China, maize morphological traits have changed with the improved genotypes over the past few decades. The two typical hybrids were compared in two consecutive years in the present study, and more maize hybrids should be evaluated to verify our results in the future.

Conclusions
Compared with Xy335, newly released maize hybrid Fm985 had a higher grain yield and a low lodging rate at a high planting density in northeast China. The improved grain yield was significantly correlated with maize kernel number per ear and lodging rate, which were directly affected by SBS and GH, as well as indirectly affected by stem lignin content and DWPU, suggesting that these characteristics which related to the genetic improvement of stalk lodging resistance, play a critical role in grain yield enhancement during modern commercial maize breeding.

Materials and methods
Site description. The study was conducted at the Jilin Academy of Agriculture Science experiment station (Gongzhuling, Jilin province, China; 43° 31′ 28″ N, 124° 48′ 35″ E) during two consecutive maize-growing seasons in 2018 and 2019. The site is located in the core zone of the "Golden corn belt" in the Songliao plain, northeast of China. This area is a classic rain-fed, long-term spring maize monoculture region with an annual rainfall of about 600 mm. The monthly temperature and precipitation during the time of the experiment were obtained from an automatic meteorological station and are shown in Fig. 7. The experimental field was a typical Black Soil (Mollisols in USDA classification) with a pH of 6.89, containing 22.01 g kg −1 organic matter, 1.75 g kg −1 total N, 17.60 g kg −1 Olsen-P, and 111.1 g kg −1 NH 4 OAc-extracted K in the 0-20 cm subsoil layer.   43 . Details about the hybrids are presented in Table 4. The experiment was arranged in a randomized complete block in a split plot arrangement and three replications, with maize hybrids in the main plots and planting density in the subplots. Maize seeds were planted using a manual seeder on May 5 in 2018 and 2019 at three planting densities, namely, 45, 000 (LD), 65, 000 (MD), and 85,000 plant/ha (HD). The sub-plots were 30 m long and 6 m wide (with 10 rows spaced 60 cm apart). To create an equal and sufficient soil growth environment, corn compound fertilizers with 220 kg N ha −1 , 100 kg P 2 O ha −1 , and 100 kg K 2 O ha −1 were applied at the sowing time according to the recommended soil nutrient management 12 . Weeds in the plots were controlled using herbicides applied before emergence (atrazine and acetochlor) and at the V6 stage (Mesotrione). Pesticides were applied as needed to control insect populations during the maize growth seasons.

Determination of plant architecture and internode morphological characteristics. At day 35
post silking, five consecutive plants in each plot were randomly selected to measure the plant architecture characteristics of plant height (PH), ear height (EH), and gravity height (GH). PH and EH referred to the distance from the ground to the top of the tassel, and to the node bearing the ear, respectively; GH was measured from the base to the fulcrum of the balance site (Fig. 8a), following the method of Xue et al. 44 .
At physiological maturity, ears were harvested from 10 m 2 plant samples at the center of each plot. Ten harvested ears were chosen to estimate the ear characteristics, including ear length, kernel number per ear, 100grain weight, and grain moisture. The maize grain yield and yield components were calculated at a moisture content of 14%.

Determination of stalk bending strength and internode morphology.
To assess maize stalk bending strength (SBS), a self-made instrument using a combination of a digital force ergometer (YYD-2; Zhejiang Top Instrument Co. Ltd) and a circle iron tool 37 was conducted at day 35 post silking 37,44 . Five adjacent plants in each plot were chosen. After removing the leaves and sheaths of each selected plant, the stems were cut at 0.8 m height from the soil surface, then were pulled until they were 30° from vertical (Fig. 9). The pulling force was determined by the ergometer, and SBS were calculated using the following formula: